Recently, a novel and highly virulent Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) emerged, causing ~40% mortality rates. MERS-CoV replicates efficiently in humans, camels and bats, but not mouse cells demonstrating a broad yet rodent-restricted host range. Ominously, person-to-person transmission and subclinical infections endanger global populations and economies. Importantly, we have demonstrated that closely related bat coronaviruses (Bt CoV), like HKU4, uses the same bat and human receptor for entry as MERS- CoV, raising the specter of further zoonotic introductions of antigenically distinct coronaviruses into human populations. Consequently, we develop a multidisciplinary program that includes X- Ray crystallography, electron microscopy, protein biochemistry, molecular virology, reverse genetics and animal model development to address fundamental questions regulating key BtCoV HKU4 and MERS-CoV-host structure-function relationships in: i) receptor mediated binding, fusion and entry, ii) cross species transmission, and iii) in vivo pathogenesis. MERS-CoV and BtCoV HKU4 encode an envelope-anchored spike glycoprotein (S) that binds to its host receptor dipeptidyl peptidase 4 (DPP4) through its S1 subunit, initiating a cascade of host serine protease regulated conformation changes that elicit S2-mediated membrane fusion and entry. The major barrier for HKU4 mediated entry into human cells occurs at protease initiated membrane fusion, rather than at the receptor binding interface. Thus, our program identifies a checkpoint associated with bat CoV cross species transmission into other mammals, including humans. In addition to identifying key structural and biochemical interactions that regulate S and DPP4 receptor ortholog-guided binding protease regulated fusion and entry, we apply reverse genetics, structure-guided mutations, and experimental evolution to study HKU4 and MERS-CoV host adaptation and pathogenesis, using recently developed transgenic animal models. In Aim 1, we study the biochemistry and structure of MERS-CoV and HKU4 bound to various species receptor-binding domains (RBD). In Aim 2, we study S processing by bat and human entry proteases. In Aim 3, we study HKU4 and MERS-CoV pathogenesis using transgenic mice and experimental evolution. The overall goal is to build a comprehensive understanding of the molecular mechanisms guiding group 2c CoV receptor recognition, entry and pathogenesis. In parallel, we produce key deliverables that are crucial for developing effective antivirals and vaccines, thereby improving rapid response and the overall global health.